Bacterial vaginosis apparatus and diagnostic methods

ABSTRACT

The present disclosure relates to the filed of medical diagnostics, specifically directed towards the field of bacterial vaginosis diagnostic methods, systems and apparatus. Also included is a multiplexed platform test for the diagnosis of infectious vaginitis.

TECHNICAL FIELD

The present invention relates generally to the field of medicaldiagnostics. More specifically, the present invention is directed towardthe field of bacterial vaginosis diagnostic methods, systems andapparatus.

BACKGROUND

The normal vaginal ecosystem is complex, predominated by Lactobacilli.Lactobacillus is estrogen dependent, maintaining a normal environmentalpH of 4.0. Lactobacillus preserves mucin gel coating over theepithelium, produces lactic acid, H₂0₂, and bacteriocins (calprotectin),and maintains an innate immune response. Lactobacillus protects againstHIV acquisition/transmission, prevents pro-inflammatory cytokines andreduces the risk of STIs (sexually transmitted infections). (Valore, AmJ Obstet Gynecol; 87: 561 (2002).)

A change in normal bacterial flora, including the reduction ofLactobacillus allows other bacteria to multiply and produce toxins whichaffect the body's natural defenses and make re-colonization of healthybacteria more difficult. Normal vaginal flora is commonly described inthe literature. Specifically normal vaginal flora is described by:Fredricks, et al., Molecular identification of bacteria associated withbacterial vaginosis. N. Engl. J. Med. 353:1899-1911. 2005; Hyman, etal., Microbes on the human vaginal epithelium. PNAS 120:7952-7957, 2005;Verhelst, et al., Cloning of 16S rRNA genes amplified from normal anddisturbed vaginal microflora suggests a strong association betweenAtopobium vaginae, Gardnerella vaginalis and bacterial vaginosis. BMCMicrobiol. 4:16. 2004; Verhelst, et al., Comparison between Gram stainand culture for the characterization of vaginal microflora: definitionof a distinct grade that resembles grade I microflora and revisedcategorization of grade I microflora. BMC Microbiol. 5:61. 2005; Zhou,et al., Characterization of vaginal microbial communities in adulthealthy women using cultivation-independent methods. Microbiology 150:2565-2573. 2004; May et al., The Identification of Vaginal LactobacillusSpecies and the Demographic and Microbiologic Characteristics of WomenColonized by These Species. The Journal of Infectious Diseases180:1950-6. 1999; and Vasquez et al., Vaginal Lactobacillus Flora ofHealthy Swedish Women. Journal of Clinical Microbiology, 40 (8).2746-2749. 2002.

Infectious vaginitis accounts for more than ten million physician officevisits each year, with approximately half of all adult women sufferingat least one episode. The three most common forms of infectiousvaginitis in decreasing incidence are bacterial vaginosis, vulvovaginalcandidiasis and trichomoniasis. Because vulvovaginal infections canresult in serious clinical sequelae, vulvovaginal symptoms and signswarrant careful evaluation and appropriate therapy.

Bacterial vaginosis (BV) is a prevalent cause of vaginal infection andis associated with several serious health conditions. BV is caused by animbalance of naturally occurring bacterial flora. BV results when thenormal, predominantly Lactobacillus, vaginal flora shifts to onedominated by Gardnerella vaginalis, Mycoplasma hominis, and a variety ofanaerobic organisms predominantly clostridial species.

Fredricks, et al. have reported considerable bacterial diversity insubjects who had BV as compared to healthy subjects, including 35bacterial phylotypes detected in samples from subjects with BV.(Fredricks, et al. “Molecular Identification of Bacteria Associated withBacterial Vaginosis” N. Engl. J. Med. 353;18 (2005).) Additionally, itwas reported that extremely high levels of bacterial DNA from severalrarely cultivated clostridial species were detected in the vaginal fluidof subjects with BV. Fielder, et al. “Changes in BV-Associated Bacterialconcentrations with Vaginal Metronicazole Therapy” ISSTDR abstract(2007)).

The pathogenesis of BV is as follows: the overgrowth of anaerobicmicroorganisms is accompanied by the production of proteolytic enzymesthat act on vaginal peptides to release several biologic products,including polyamines, which volatize in the accompanying alkalineenvironment to elaborate foul-smelling trimethylamine. Polyaminesfacilitate the transudation of vaginal fluid and exfoliation ofepithelial cells, creating a copious discharge. Clue cells are formedwhen such organisms are present in high numbers, adhere to exfoliatedepithelial cells in the presence of an elevated pH. (Sobel, J D, N Engl.J. Med. 337: 1896-1903 (1997).)

Untreated BV may cause serious complications, such as increasedsusceptibility to sexually transmitted infections and may present othercomplications for pregnant women. BV has also been associated with anincrease in the development of Pelvic Inflammatory Disease followingsurgical procedures such as a hysterectomy or an abortion. BV can becured by antibiotics such as metronidazole and clindimicyn. Thus it isimportant to have an effective efficient method for diagnosis.

Prior BV diagnostics focus on causative agents, either applying director indirect measures. These diagnostics include detection of Gardnerellavaginalis through a nucleic acid based test. Additionally, priordiagnosis of BV includes detection of elevated enzymes such as sialidaseactivity, an enzyme produced by bacterial pathogens associated with BVincluding Gardnerella, Bacteroides, Prevotella and Mobilincus.

All of the references cited in this disclosure are hereby incorporatedin their entirety.

SUMMARY OF THE DISCLOSURE

The present disclosure teaches a method for the diagnosis of BV througha quantitative determination of Lactobacillus, establishing a measure ofclinically relevant Lactobacillus in healthy vaginas, and correlating adiminished amount of Lactobacillus with the diagnosis of BV. The presentdisclosure also teaches a method for detecting the occurrence ornon-occurrence of bacterial vaginosis in a patient comprising the stepsof collecting a biological sample from a patient, quantitativelymeasuring the presence of Lactobacillus; and correlating a diminishedpresence of Lactobacillus with the diagnosis of bacterial vaginosis. Thedisclosure further teaches using quantitative polymerase chain reactionto Lactobacillus in order to quantitate the amount of Lactobacillus. Thedisclosure further teaches using any analyte sandwich assay (i.e.lateral flow immunoassay, ELISA or direct DNA detection) to quantitatethe amount of Lactobacillus. The disclosure further teaches a method fordiagnosis of BV, Candida and Trichomonas utilizing lateral flowimmunoassay to quantitate Candida, Trichomonas and Lactobacillus in amultiplexed assay.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphic representation of an inverse relationship betweenLactobacillus crispatus and BV pathogens.

FIG. 2 is a graphic representation of changes in BV flora followingMetronidazole treatment.

FIG. 3 is a flow chart representation of a syndromic approach todiagnosis of BV, Candida and Trichomonas, collectively called vaginosis.

FIG. 4 is a flow chart representation of an additional step in thesyndromic approach to diagnosis of BV, Candida and Trichomonas,collectively called vaginosis.

DETAILED DESCRIPTION OF THE INVENTION

Lactobacillus crispatus, is the predominant Lactobacillus speciespresent in “healthy vaginas” as shown in the control subjects ofTable 1. Table 1 includes the results of a study of the bacteriaidentified by broad-range 165 rDNA polymerase chain reaction in vaginalfluid from subjects with bacterial vaginosis. (Fredricks et al. N Eng JMed 2005) There are a large and diverse number of bacterial speciesfound in the vagina of subjects with BV. FIG. 1 illustrates theexistence of an inverse relationship between Lactobacillus crispatus andBV pathogens in subjects with BV. Thus the level of Lactobacilluscrispatus significantly decreases with a diagnosis of BV.

TABLE 1 Bacteria Identified by Broad-Range 16SrDNA Polymerase ChainReaction in Vaginal fluid from Subjects with and without BacterialVaginosis. Subjects with BV Control Subjects without BV 1 2 3 4 5 6 7 89 1 2 3 4 5 6 7 8 Percentage of Clones Lactobacillus crispatus 49 74 9948 60 89 40 100 L. jensenii 2 1 L. gallinarum 13 L. gasseri 9 1 L.vaginalis 2 Staphylococcus 2 epidermidis S. lugdunensis 1 Clostridium 3perfringens (96%) AB045286 Ureaplasma parvum 1 L. iners 7 22 3 1 5 38 146 36 10 60 Gardnerella vaginalis 35 13 2 4 28 1 25 31 39 14 UnculturedAB034121 43 66 34 36 17 26 (90.6%) (BVAB1) Uncultured AF407407 10 4 10 35 4 4 (90.9%) (BVAB2) Uncultured 1 1 1 ULO278163 (92.9%) (BVAB3)Atopobium vaginae 5 3 21 1 3 3 11 11 Leptotrichia amnionii 6 2 8 1 3 1010 Megasphaera elsdenii 4 10 7 1 18 3 2 13 6 (93.8%) AY038994 M.micronuciformis 1 (94.6%) AF473833 Eggerthella 2 2 2 1 4 1 3 8hongkongensis (91.8%) AY288517 Porphyromonas 2 5 asaccharolyticaDialister species (a) 1 1 2 2 1 Dialister species (b) 1 2 2 2 1 (94.8%)AF371693 Sneathia sanguinegens 3 2 16 9 9 Prevotella genogroup 21 24 1 79 12 20 1 Prevotella genogroup 7 4 6 7 1 2 Prevotella genogroup 7 3 3 P.bivia P. buccalis 6 P. dentalis (93.2%) 2 2 X81876 P. disiens P. oulorum(90.6%) L16472 P. shahii (90.7%) AB108825 Uncultured 4C28d-23 1 3 1 2(91.2%) AB034149 Candidate division 2 2 8 TM7 (93.7%) AF125206Mobiluncus mulieris Peptoniphilus 1 lacrimalis Peptoniphilus species 1 2Peptostreptococcus 3 2 1 micros (97.8%) AF542231 Gemella bergeriae 1(95.8%) Y13365 Aerococcus species 1 2 Anaerococcus tetradius Uncultured(89.8%) AF371910 Uncultured (88.4%) AJ400235 Veillonella species 1

As shown in FIG. 2, Polymerase Chain Reaction (PCR) was used to surveyBV flora before and after metronidazole treatment. (Ferris et al. J. ofClin. Microbiology, 2007). The species composition for patients prior totreatment was variable. Lactobacillus (in particular Lactobacillusinners) was prominent in all patients post-treatment. Atopobium vaginaeconcentrations were highest for patients who failed or respondedincompletely to treatment and lowest for patients who were cured. Thusthe results illustrated that post treatment, Lactobacillus predominate.Thus, a normal level of Lactobacillus present in the vaginal flora iskeystone to a “healthy vagina”.

The present disclosure teaches a method for the diagnosis of BV.Quantitative measure of Lactobacillus as a measure of a healthy vaginaand an inverse correlation with BV yields a diagnostic test for BV. Thismethod differs from previous diagnostics where the presence of themultitude of agents associated with BV, such as Gardnerella, indirectenzyme measures, Amsel criteria etc., was identified for diagnosis. Thepresent disclosure teaches quantitatively measuring clinically relevantLactobacilli. These Lactobacilli include, but are not limited to,crispatus, jensenii, and gasseri, either independently or incombination. Quantitative measurement can be achieved by a variety ofmethods known in the art. These methods include, but are not limited to:antibody/homologous cross reactive but specific reagents, quantitativepolymerase chain reaction, and any “sandwich” assay—both antibody andnucleic acid based. Further included are direct antigen measurement andlateral flow immunoassay including utilizing SERS technology. Thepresent disclosure teaches establishing a clinically relevant cut offvalue for the Lactobacillus measure from clinical trials and the use ofstatistical measures of relevance (ROC curves (receiver operator curves)as would be understood by one of skill in the art.

Table 2 lists the analyte targets, antibodies for cytoplasmic proteins,antibodies for surface proteins and sequence information for applicabletargets, including Candida, Trichomonas and Lactobacillus. Using thedisclosed sequences, one with skill in the art can readily and withoutundue experimentation, develop antibodies to Candida, Trichomonas andLactobacillus. As such, antibodies for Candida and Trichomonas arealready known and listed in the table. Additionally, methods to developantibodies using sequence derived antigens as described can be found atStrategic Diagnostics Inc, http://www.sdix.com/. In the case ofLactobacillus, the sequences disclosed and known are used to determineantigenic regions and will be further used to raise antibodies.

TABLE 2 Antibody for Analyte Antibody for cytoplasmic surface Targetsprotein protein Sequence information Candida PA1-7206 Polyclonal,MA1-7009, The completed assembly #21 contains 15.845 Mb of albicansABR—Affinity BioReagents Monoclonal, DNA, organized into the 8 C.albicans chromosomes. ab53891 Polyclonal, Abcam ABR—Affinity The geneand protein sequences or the sequence of part Ab34211 monoclonal, AbcamBioReagents or all of a chromosome can be retrieved from the 1.monoclonal, Candida Genome AbD serotecDatabase.http://www.candidagenome.org/ 2. monoclonal, * The genomicsequence of Candida glabrata AbD serotec chromosomes A-M is available inGenebank. 3. monoclonal,http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?taxid= AbD serotec284593&chr=A 1750-5007 Polyclonal, AbD serotec Trichomonas 9155-9529monoclonal, C65101M The Trichomonas vaginalis G3 whole genome shotgunvaginalis AbD serotec monoclonal, (WGS) project has the acession# Thecell localization of the Meridian NZ_AAHC00000000. This version of theproject(01) immunogen for this antibody C65657M has the accession#NZ_AAHC01000000, and consists is unknown. monoclonal, of sequencesNZ_AAHC01000001- Meridian NZ_AAHC01075128. C65675M Length: 178,315,610bp (unfinished). monoclonal, Meridian C65526M monoclonal, Meridian PTV65 polyclonal, HyTest Ab24317 Polyclonal, abcam Lactobacillus 146nucleotide sequences in CoreNucleotide Database, crispatus for example:gene for 16S rRNA, glyceraldehyde-3- phosphate dehydrogenase, S-layerproteins and 60 kDa chaperonin. 63 protein sequences, for example:phenolic acid decarboxylase, alpha-enolase, transposase, truncated PTSEIIC-like protein and surface layer protein. Lactobacillus 37 nucleotidesequences in CoreNucleotide Database, jensenii for example: gene for 16Sribosomal RNA, rpoA gene for RNA polymerase alpha subunit, pheS gene forphenylalanyl-tRNA synthase alpha subunit, ptsH gene, promoter region and5′ UTR, rpsU gene, promoter region and 5′ UTR, 60 kDa heat shockprotein, recA gene for recombinase A, mRNA for putative elongationfactor Tu, 60 kDA chaperonin and 16S-23S rRNA intergenic spacer regionand 23S rRNA gene 5' flanking region. 6 protein sequences, for example:RNA polymerase alpha subunit, phenylalanyl-tRNA synthase alpha subunit,60 kDa heat shock protein, recombinase A, putative elongation factor Tuand 60 kDa chaperonin. Lactobacillus 261 nucleotide sequences inCoreNucleotide Database, gasseri for example: gene for 16S ribosomalRNA, 60 kDa chaperonin, rpoA gene for RNA polymerase alpha subunit, pheSgene for phenylalanyl-tRNA synthase alpha subunit, putative complementfactor, acidocin LF221B, and putative immunity protein genes, recA genefor recombinase A, pbgal gene for phospho-beta- galactosidase, ATPsynthase beta chain, aggregation promoting factor, gassericin T generegion, Gassericin A, phospho-beta-galactosidase and many sequences frompatents. Genome sequences for Lactobacillus gasseri ATCC 33323, Length:1,894,360 bp.3967 protein sequences, for example: putative immunityprotein, acidocin LF221A, putative complemental factor, Lysin, Holin,phospho-beta-galactosidase, gassericin K7 B, putative ATP-dependenttransport protein, aminopeptidase N, putative branched-chain amino acidtransporter, beta- glucuronidase, putative regulatory protein,gassericin T, Gassericin A, ATP synthase alpha subunit, RNA polymerasealpha subunit, Ribosomal protein L34, RNase P protein component,Preprotein translocase subunit YidC, Predicted membrane protein,transcriptional regulator, Aggregation promoting factrelated surfaceprotein and uncharacterized conserved secreted or membrane protein

The present disclosure also incorporates by reference the technologydisclosed for the use of SERS tags in assays including sandwich assaysand lateral flow immunoassays (LFI). In particular, the followingpatents and pending applications are incorporated herein by reference;U.S. Pat. No. 6,514,767, Surface Enhanced Spectroscopy—Active CompositeNanoparticles; U.S. Pat. No. 7,192,778, Surface EnhancedSpectroscopy—Active Composite Nanoparticle; U.S. Patent App. PublicationNo. US-2005-0219509; Surface Enhanced Spectroscopy—Active CompositeNanoparticles; PCT Patent App. No. PCT/US07/61136 (unpublished), LateralFlow Immunoassay with Encapsulated Detection Modality; PCT PatentPublication No. WO-2007/092941, SERS Nanotag Assays.

SERS allows detection of molecules attached to the surface of a singleRaman-enhancing nanoparticle. A Raman-enhancing metal that hasassociated or bound to it a Raman-active molecule(s) is referred to as aSERS-active nanoparticle. Such SERS-active nanoparticles can haveutility as optical tags. For example, SERS-active nanoparticles can beused in immunoassays when conjugated to an antibody against a targetmolecule of interest. If the target of interest is immobilized on asolid support, then the interaction between a single target molecule anda single nanoparticle-bound antibody could be detected by searching forthe Raman-active molecule's unique Raman spectrum. Furthermore, becausea single Raman spectrum (from 100 to 3500 cm⁻¹) can detect manydifferent Raman-active molecules, different SERS-active nanoparticlescan be used in multiplexed assay formats. In one embodiment, thedisclosure provides for a single diagnostic for BV through themeasurement of Lactobacillus. In an alternative embodiment, thedisclosure provides for a multiplexed assay for BV, Candida, andTrichomonas, determining BV through the measurement of Lactobacillus.

The present disclosure also provides a lateral flow immunoassay (LFI)featuring encapsulated metal particles. Briefly, the encapsulatedparticles may use SERS nanotags as the detection modality. The use ofencapsulated particles as a detection modality, in particularencapsulated SERS tags increases the sensitivity of an LFI prepared forvisual reading and introduces the ability to obtain substantially moresensitive qualitative results or quantitative results through theanalysis of a SERS spectrum read from an LFI prepared in accordance withthe present embodiment. The use of SERS as detection modality alsoenhances the ability of an LFI device to be used for a multiplexed test.Other embodiments include LFI devices specifically configured to testvaginal samples, a reader for the detection and interpretation of amultiplexed assay and the hardware and software components used toimplement the reader.

Note that throughout this application various citations are provided.Each of these citations is specifically incorporated herein by referencein its entirety.

EXAMPLE 1

In one embodiment, Lactobacillus sequence will be used to determineantigenic regions and use this to raise antibodies. These antibodieswill be used for quantitative measurements of Lactobacillus.

EXAMPLE 2

In another embodiment, the sequences known for Lactobacillus are used toperform quantitative polymerase chain reaction for a determination ofthe amount of Lactobacillus.

EXAMPLE 3

In one embodiment, a quantitative measurement of clinically relevantLactobacilli is performed. This may include crispatus, jensenii, gasserieither independently, or with antibody/homologous cross reactive butspecific reagents. As is known by one with skill in the art, aclinically relevant cut off value for the Lactobacillus measure viaclinical trials and the use of statistical measures of relevance (i.e.ROC curves) are developed. Once the clinically relevant cut off valuefor Lactobacilli is established, a diagnostic test is performed, whereinthe Lactobacilli of a patient is measured, and a diagnosis of BV isestablished based on a low clinically relevant Lactobacilli value.

EXAMPLE 4

In one embodiment, the disclosure teaches a syndromic approach tovaginitis diagnosis. The three most common forms of infectious vaginitisin decreasing incidence are bacterial vaginosis, vulvovaginalcandidiasis and trichomoniasis. As shown in FIG. 3, incorporating thethree measures in one test 300 results in a very powerful test. Candidaand Lactobacillus measures 302(a), 302(b), 304(a) and 304(b),respectively, are quantitative while the Trichomonas would be a testbased on the presence or absence of Trichomonas 306(a), 306(b). As isknown by one with skill in the art, a clinically relevant cut off valuefor the Lactobacillus measure via clinical trials and the use ofstatistical measures of relevance (i.e. ROC curves) are developed. Oncethe clinically relevant cut off value for Lactobacilli is established, adiagnostic test is performed, wherein the Lactobacilli of a patient ismeasured, and a diagnosis of BV is established based on a low clinicallyrelevant Lactobacilli value. Similarly, for vulvovaginal candidiasis,clinical trials and the use of statistical measures of relevance (i.e.ROC curves) are developed. Once the clinically relevant cut off value isdeveloped for Candida, diagnosis of vulvovaginal candidiasis isestablished based on a high clinically relevant Candida value.Trichomoniasis is diagnosed based on the presence of Trichomonas. In oneembodiment a kit comprising the three tests based on LIF is envisionedusing SERs tags for the relevant moieties. As shown in FIG. 4, anadditional step 308 may selectively be implemented where analytes forcausative agents to increase clinical sensitivity and specificity areadded to the initial quantitative lactobacillus determination.

While the invention has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the invention and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims.

1. A method for detecting the occurrence or non-occurrence of bacterialvaginosis in a patient comprising the steps of: collecting a biologicalsample from a patient; quantitatively measuring the presence ofLactobacillus; and correlating the diminished presence of Lactobacilluswith a diagnosis of bacterial vaginosis.
 2. The method of claim 1wherein said step of quantitatively measuring the presence ofLactobacillus comprises performing polymerase chain reaction forLactobacillus.
 3. The method of claim 1 wherein said step ofquantitatively measuring the presence of Lactobacillus comprisesperforming a sandwich assay for Lactobacillus.
 4. The method of claim 3wherein said sandwich assay is a lateral flow immunoassay.
 5. The methodof claim 4 wherein said lateral flow immunoassay comprises SERStechnology.
 6. The method of claim 1 wherein said Lactobacillus isselected from the group consisting of Lactobacillus crispatus,Lactobacillus jensenii and Lactobacillus gasseri.
 7. A method for thediagnosis infectious vaginitis comprising collecting a biological samplefrom a patient; measuring the presence of Lactobacillus, Candida andTrichomona; and correlating said measurements; wherein a diminishedpresence of Lactobacillus is indicative of bacterial vaginosis, anincreased presence of Candida is indicative of vulvovaginal candidiasisand the presence of Trichomona is indicative of trichomoniasis.
 8. Themethod of claim 7, wherein the measuring is performed in a single testkit.
 9. The method of any of claims 7 or 8, wherein the measuring ofLactobacillus and Candida are quantitative measurements.
 10. The methodof any of claims 7 or 8, wherein said measuring is for the presence orabsence of Trichomona.
 11. The method of any of claims 7 or 8, whereinsaid measuring is performed comprising a lateral flow immunoassay.
 12. Akit comprising an apparatus and instructions wherein the apparatus is amultiplex platform wherein testing for Lactobacillus, Candida andTrichomona is performed.
 13. The method of claim 9, wherein saidmeasuring is performed comprising a lateral flow immunoassay.
 14. Themethod of claim 10, wherein said measuring is performed comprising alateral flow immunoassay.